Most wild species of cats produce mis-shapened sperm cells in their semen, a condition called teratospermia. We are interested in both the cause and significance of this finding. Male cats that ejaculate many sperm with abnormal shapes also produce normal appearing sperm cells that often cannot fertilize eggs. New studies have discovered that the defects occur at the subcellular level. These sperm have problems in undergoing capacitation and the acrosome reaction, two important processes that are necessary for fertilization to occur. Sperm from these males also have problems undergoing the necessary changes to proteins on the sperm head that allow them to bind and penetrate eggs. These findings are important for several reasons. They allow us to identify the precise sites of infertility, so we can develop methods to circumvent such problems. This past year we developed a technique, "intracytoplasmic sperm injection," to place sperm from teratospermic males directly into the egg using a micropipette injection system. These sperm still have problems completing fertilization. These males have inherent defects that present serious challenges to effective reproduction. An important reason to continue such studies is that men also tend to produce many abnormal sperm. It may well be that what we learn from the cats will provide new clues about solving human infertility problems. Our most recent discovery is that sperm DNA from teratospermic cats appears to be unstable, a problem that may occur as the sperm are growing and maturing in the testis. Studies are in progress to measure the amount of protamines in male cats that produce mostly normal versus abnormal sperm. The hope is that we can better understand this condition and perhaps develop a treatment. Our laboratory has long promoted genome resource banking, the organized collection, storage and use of sperm, embryos, tissues, blood products and DNA. We especially are interested in developing methods that would allow the better management of endangered cat species using artificial insemination with cryopreserved sperm. Currently only about 30% of cat sperm survive freezing and storage in liquid nitrogen. Solving this problem is not simple because successful cryopreservation requires a thorough understanding of the optimal medium and cryoprotectant and the ideal rate to cool, freeze and thaw sperm. We are reassessing and modifying existing technology. Male cats that ejaculated the most mis-shapened sperm suffered the most damage during cooling, revealing yet another disadvantage for males suffering from teratospermia. Damage could be reduced, however, if the rate of sperm cooling was slowed. Studies in progress focus on the thermal properties of the sperm membrane. We believe that understanding the plasma membrane is a logical first step to eventually improving our technical ability to fully protect sperm during the stresses of freezing and thawing. To be successful, artificial insemination must be performed at the appropriate time during a female cat's cycle. The ovaries need to be quiescent before hormones are injected to stimulate ovulation. In an effort to suppress the ovaries before giving hormonal therapy, we are testing the usefulness of the pineal hormone, melatonin. Once "shut- down" by melatonin, the ovary should respond more effectively to hormone treatments used to stimulate the follicles to grow and ovulate. A study was conducted to determine how much oral melatonin to give by gelatin capsule. Oral melatonin at 30 mg/day suppressed follicle activity and cats quickly returned to estrus within a couple of weeks after treatment was stopped. Our eventual objective is to develop a safe and reliable treatment that will increase the success of using artificial insemination in endangered cats. In vitro fertilization and embryo transfer could be useful for assisting in the genetic management of endangered cat populations. However, so far, pregnancy success has been low using these techniques. We believe this is because the embryos are poor quality or the uterine environment is not ideal in the host mother. For embryo transfer, the recipients usually are treated with the two hormones, eCG to induce follicles to grow on the ovary, and hCG to produce ovulation. In a recent study, the endocrinology and uterine histology of domestic cats treated with several different hormone treatments was evaluated. Females treated with the usual eCG-hCG treatment frequently experienced abnormal characteristics. However, cats that were in natural heat and induced to ovulate with gonadotropin-releasing hormone (GnRH) had a much more normal maternal environment. We evaluated the suitability of cats treated with GnRH as recipients to nurture embryos produced by in vitro fertilization. Compared to embryos that were cultured in the laboratory, embryos placed into GnRH-treated cats developed well in the uterus. Half of these cats became pregnant with more than half the embryos surviving to implant into the uterus. This was a much higher rate of success than we have achieved previously. Our findings indicate that most embryos produced by in vitro fertilization are normal and can develop after transfer to the recipient. Studies in progress are assessing the ability of GnRH-treated recipients to carry these implanted embryos to term and result in healthy young. We have also been exploring techniques that might eventually allow the production of genetically identical cats. Such animals would be especially important in the study of certain infectious viruses which can kill both domestic and endangered cat species. Methods have been tested to mechanically bisect cat embryos. But, an efficient procedure seems to involve the chemical disruption of the individual cells comprising the embryo. Once loosened from the embryo, these cells appear able to grow into separate embryos in the laboratory; genetic clones. Future studies will focus on growing these embryos to later stages of development before attempting embryo transfer. By having identical embryos we also predict the more rapid development of better methods for culturing embryos before they are transferred to surrogate mothers.